This research was supported by NIH-CA100324, Einstein National Cancer Institute's cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.

All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
1. Generating Fluorescently Labeled Mouse Model and Tumor Cells
<ol>
	<li>Prepare 100 ml of 0.1 % (w/v) bovine serum albumin/phosphate buffered saline (BSA/PBS) buffer by mixing 0.1 g of BSA with 100 ml of PBS.</li>
	<li>Prepare fluorescent...

The authors have nothing to disclose.

High resolution in vivo optical imaging combined with fluorescently labelled functional tags such as proteins and antibodies has dramatically increased our understanding of the metastatic cascade. It has enabled direct visualization and quantification of single-cell and sub-cellular parameters in tumor cells, host cells and their microenvironment. This imaging within the primary tumor has led, for example, to the discovery of discrete microenvironments that are supportive of either growth invasion or disseminati...

High resolution intravital optical imaging has proven to be crucial to understanding many biological processes, allowing single-cell and sub-cellular parameters to be measured and quantified. In cancer research, intravital imaging of tumor and stromal cells has led to the discovery of many microenvironmental interactions 6-11 that are only present in the intact animal.
Discoveries about microenvironments associated with intravasation and dissemination of tumor cells in breast cancer using single cell resolution optical imaging in vivo has even led to novel markers for prognosis and response to treatment in breast cancer patients 12-16. The best imaging technologies available for viewing deep within intact internal vital organs are the clinical modalities (MRI, PET, CT) which offer excellent views of the entire organ and can reveal pathologies even before they produce clinical symptoms. They are unable, however, to reveal the earliest stages of metastasis and the cellular mechanisms driving tumor progression due to their lack of single cell resolution. By the time lung metastases are visible in these modalities, they are well established and proliferating. Given the estimate that 90% of disseminated tumor cells that arrive to the lung either do not survive 17 or initially remain dormant 18, and the observation that they arrive much earlier than previously expected 19, imaging the earliest steps of arrival and survival becomes crucial to understanding the process of metastatic seeding and recurrence of tumor growth at distant sites.
Performing these observations in the lung has proven extremely difficult however; the vast majority of imaging studies have utilized ex vivo or explant preparations 20-23, which only give a view into the lung at single time points. While these preparations do provide useful information, they do not give a complete understanding of the interactions, cause and effect relationships, and dynamics that occur between the various components of the microenvironment. The lack of a proper circulatory system (and concomitant imbalance of homeostasis) and the disconnection from the rest of the body&#39;s immune system makes it desirous to validate the conclusions that these preparations generate in intact tissue in vivo.
Many groups have performed intravital imaging of the intact lung 2,4,5,24-33 with Wearn and German being the first to surgically expose the pleural layer 24 and Terry the first to utilize an implantable imaging window 25.
High resolution imaging in the lung is greatly hindered by the lung&#39;s constant motion and several techniques have been developed to overcome this limitation. Wagner and Filley 27 studied the natural motion of the canine lung and designed their surgical protocol to locate their implanted window over a relatively stationary region while Wagner utilized vacuum in his window surgical preparation to immobilize the tissue 28. Since that time, a variety of techniques have been utilized to image the lung including: bronchus clamping, sequential apnea and gated imaging, oversampled acquisition, gluing of the lung lobe and vacuum 34. Each of these has its advantages and disadvantages and no one technique has emerged as superior to another 34. For example, bronchus clamping and sequential apnea alter the normal exchange of gases in the lung and may cause atelectasis. Gated imaging and oversampled acquisition do not suffer from these disadvantages but require high-speed or specialized imaging equipment not widely accessible. Finally both gluing of the lung and the vacuum technique avoid both of the above mentioned drawbacks, but may exhibit shear force induced injury if care is not taken. In recent years, the vacuum window has been miniaturized and adapted for use in mice using confocal and multiphoton microscopy 4,5,33 and excellent high-resolution imaging has been attained 2. Table 1 summarizes this rich history and highlights those papers which describe novel advancements in the use of intravital lung imaging windows.
This protocol describes the use of extended time-lapse multiphoton intravital microscopy to image metastasis in the live, intact lung with the highest subcellular resolution possible. Images are acquired for up to 12 hr using a multiphoton microscope equipped with a high numerical aperture objective lens and multiple photomultiplier tube (PMT) detectors. Transgenic mouse models are utilized to fluorescently label native macrophages along with fluorescent high molecular weight dextran and fluorescent protein transfected tumor cells (to label the vasculature and tumor cells respectively). While this choice of fluorescently labeled cells enables visualization of tumor cell-endothelial cell-macrophage interactions and dynamics, this protocol will work for any strain of fluorescent or non-fluorescent mouse. After acquisition, residual drift motion (if any) is eliminated using a Fiji plugin 35,36 and custom macros time average the vascular channel to eliminate flashing caused by unlabeled circulating blood cells.
While this protocol focuses on imaging metastasis, the techniques are applicable to many other biological processes observable with high-resolution single-cell imaging in the lung.

<table border="0" cellpadding="0" cellspacing="0" width="1579">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Nickel-Plated Brass Vacuum Regulator 1/8 NPT Female, w/ Gauge, 0 - 20&#34; Hg Vacuum</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">4172K12&#160;</td>
			<td style="width:223px;">Vacuum Regulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Brass Barbed Hose Fitting Adapter for 1/4&#34; Hose ID X 1/8&#34; NPTF Male Pipe</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5346K13</td>
			<td>Vacuum Regulator Hose Adapter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Pyrex Brand Filtering Flasks with Tubulation; Neck tooled for rubber stopper No. 4; Capacity: 50mL</td>
			<td style="width:291px;">Corning Life Sciences Glass</td>
			<td style="width:141px;">5360-50</td>
			<td>Vacuum Flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Round Glass Coverslips Thickness #1.5, 0.16-0.19mm 10mm dia.&#160;</td>
			<td style="width:291px;">Ted Pella, Inc.</td>
			<td>260368</td>
			<td>Cover slips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Exel International Disposable Safelet I.V. Catheters; 22gx1 in.&#160;</td>
			<td style="width:291px;">Exel International</td>
			<td style="width:141px;">26746</td>
			<td>Tracheal Catheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0</td>
			<td style="width:291px;">VWR</td>
			<td style="width:141px;">95056-992</td>
			<td>String</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Liquid Super Glue, Clear, 0.14oz</td>
			<td style="width:291px;">Hendel Corp.</td>
			<td style="width:141px;">LOC1647358</td>
			<td>Cyano-acrylate Glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Tetramethylrhodamine isothiocyanate&#8211;Dextran</td>
			<td style="width:291px;">Sigma-Aldrich</td>
			<td style="width:141px;">T1287-500MG</td>
			<td>155kD Dextran</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laboratory Clear Tygon PVC Tubing, 1/16&#34; ID, 1/8&#34; OD, 1/32&#34; Wall Thickness, 25 ft. Length</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5155T12</td>
			<td>Thin Tubing &#38; Tubing for Luer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Crack-Resistant Polyethylene Tubing, 1/8&#34; ID, 1/4&#34; OD, 1/16&#34; Wall Thickness, White, 50 ft. Length&#160;</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5181K24&#160;</td>
			<td>Thick Tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Depillatory Lotion</td>
			<td style="width:291px;">Nair</td>
			<td style="width:141px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Micro Medical Tubing 95 Durometer LDPE</td>
			<td style="width:291px;">Scientific Commodities Inc.</td>
			<td style="width:141px;">BB31695-PE/1</td>
			<td>Tubing for tail vein catheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">30 G x 1 in. BD PrecisionGlide Needle</td>
			<td style="width:291px;">BD</td>
			<td style="width:141px;">305128</td>
			<td>Needles for tail vein catheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Puritan Nonsterile Cotton-Tipped Swabs&#160;</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td>867WCNOGLUE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32&#34; x 1/16&#34; Tube ID</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5117k51</td>
			<td>Connectors between tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">One-Hole Rubber Stoppers</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td>14-135F</td>
			<td>Stopper for Vacuum Flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">SHARP Precision Barrier Tips, For P-100, 100&#181;l</td>
			<td style="width:291px;">Denville Scientific Inc.</td>
			<td style="width:141px;">P1125</td>
			<td>Pipette Tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laboratory tape</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">159015R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Puralube</td>
			<td style="width:291px;">Henry Schein Animal Health</td>
			<td style="width:141px;">008897</td>
			<td>Opthalmic Ointment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Gemini Cautery Kit</td>
			<td style="width:291px;">Harvard Apparatus</td>
			<td style="width:141px;">726067</td>
			<td>Cautery Pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4&#34; Length</td>
			<td style="width:291px;">Roboz Surgical</td>
			<td style="width:141px;">RS-5135&#160;</td>
			<td>Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Extra Fine Micro Dissecting Scissors 4&#34; Straight Sharp/Sharp 24mm</td>
			<td style="width:291px;">Roboz Surgical</td>
			<td style="width:141px;">RS-5912</td>
			<td>Sharp Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Micro Dissecting Scissors 4&#34; Straight Blunt/Blunt</td>
			<td style="width:291px;">Roboz Surgical</td>
			<td style="width:141px;">RS-5980</td>
			<td>Blunt Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Wipes</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">06-666-A&#160;</td>
			<td>Harness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">PhysioSuite System</td>
			<td style="width:291px;">Kent Scientific</td>
			<td style="width:141px;">PhysioSuite</td>
			<td>Vitals Monitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">1 mL Syringe, Tuberculin Slip Tip</td>
			<td style="width:291px;">BD</td>
			<td style="width:141px;">309659</td>
			<td>Syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Cyano acrylate</td>
			<td style="width:291px;">Staples</td>
			<td>LOC1647358</td>
			<td>Cover Slip Adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Petroleum Jelly</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">19-086291</td>
			<td>Water Barrier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Adapter Luer Cannulla 1.5-2.2mm</td>
			<td style="width:291px;">Harvard Apparatus</td>
			<td style="width:141px;">734118</td>
			<td>Catheter Connector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">MouseOx oximeter, software and sensors</td>
			<td style="width:291px;">STARR Life Sciences</td>
			<td style="width:141px;">MouseOx</td>
			<td>Pulse Oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Isoethesia (isoflurane)</td>
			<td style="width:291px;">Henry Schein Animal Health</td>
			<td style="width:141px;">50033</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Oxygen</td>
			<td style="width:291px;">TechAir</td>
			<td style="width:141px;">OX TM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">1 x PBS</td>
			<td style="width:291px;">Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">PVC Ball Valve, Push to Connect, 1/4 In</td>
			<td style="width:291px;">Grainger</td>
			<td>3CGJ7</td>
			<td>Vacuum Valve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Small Animal Ventilator</td>
			<td style="width:291px;">Harvard Apparatus</td>
			<td style="width:141px;">683</td>
			<td>Alternative is available from Kent Scientific: MouseVent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">OptiMEM Reduced Serum Medium</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td style="width:141px;">31985062&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td style="width:141px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice</td>
			<td style="width:291px;">Jackson Laboratory</td>
			<td style="width:141px;">026051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Multiphoton Microscope</td>
			<td style="width:291px;">Olympus</td>
			<td style="width:141px;">Fluoview FV1000</td>
			<td>Alternative to custom built scope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Environmental Enclosure</td>
			<td style="width:291px;">Precision Plastics</td>
			<td style="width:141px;">Chamber for FV1000</td>
			<td>Alternative to custom built enclosure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Phosphate Buffered Saline</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laser Power Meter</td>
			<td style="width:291px;">Coherent</td>
			<td style="width:141px;">FieldMaxIITOP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laser Power Meter Head</td>
			<td style="width:291px;">Coherent</td>
			<td style="width:141px;">PM10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">pcDNA3-Clover Fluorescent Protein Vector</td>
			<td style="width:291px;">Addgene</td>
			<td style="width:141px;">40259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">G418 Sulfate Selective Antibiotic</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td style="width:141px;">10131027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">MoFlo Fluorescent-Activate Cell Sorter&#160;</td>
			<td style="width:291px;">Beckman Coulter</td>
			<td style="width:141px;">XDP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Trypsin EDTA 1X</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">25-052-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">40 &#181;m Mesh</td>
			<td style="width:291px;">Falcon</td>
			<td style="width:141px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">96 Well Plate</td>
			<td style="width:291px;">Costar</td>
			<td style="width:141px;">3599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">60 mm Culture Dish</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">430196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">10 cm Culture Dish</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Bovine Serum Albumin</td>
			<td style="width:291px;">Sigma-Aldrich</td>
			<td style="width:141px;">A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Dulbecco&#39;s Phosphate Buffered Saline 1X</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">C57BL/6J Mouse</td>
			<td style="width:291px;">Jackson Laboratory</td>
			<td style="width:141px;">000664&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Kim Wipes</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">06-666-A&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

To demonstrate the type of results that can be achieved with this method, we injected E0771-LG tumor cells labeled with the fluorescent protein Clover into the tail vein of MacBlue mice 44 at varying time points before surgery. After surgery, 155 kD rhodamine labeled dextran was injected IV to mark the vasculature and time-lapse imaging was performed.
When imaging mice 24 hr post injection, single cells are visible in...

Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

The overall goal of this method is to perform long-term, high-resolution, single-cell imaging of the intact lung using a vacuum stabilized imaging window. This method can help answer key questions in the field of cancer research;such as, what interactions secure between stroma and tumor cells during the earliest steps of metastasis. The main advantage of this technique is that the live, intact lung can be theorically visualized at a single-cell resolution for up to 12 hours.Begin by tying a three inch length of 2-0 suture to a tracheal catheter, one quarter inch above the bushing, with a double-knot. Then apply eye ointment to an anesthetized mouse's eyes and use depilatory cream to remove the hair from the left side of the animal from the mid-line of the chest to a quarter of the back and from the axilla to just under the rib cage. Clean any excess lotion and sterilize the exposed skin with 70%ethanol.Next, intubate the animal. And tie the 2-0 suture around the snout, under the front teeth. Move the animal to the surgical area, taking care not to dislodge the catheters, and turn on the ventilator.Connect the ventilator to the tracheal catheter. Then tape the catheter to the snout and the left forelimb to the catheter. After confirming a lack of response to toe-pinch, use sharp scissors to remove one square centimeter of skin above the left chest wall.Lift the mammary fat pad and cauterize any exposed blood vessels. Next, resect the fat pad and remove the muscle layer down to the rib cage, taking care not to cut the axillary vein at the base of the forelimb. Use forceps to lift the sixth rib.Then, holding the sharp scissors at a shallow five degree angle, cut the rib near the edge of the opening in the skin. Remove four consecutive ribs to widen the opening in the chest wall, exposing the entire lung lobe. Then carefully transfer the mouse onto the microscope imaging stage.With the vacuum off, fill the inner chamber of the vacuum window with PBS. Invert the mouse and position the exposed lung over the vacuum imaging coverslip window. Slowly, turn on the vacuum.Place a restraining harness, made of tissue paper folded in half, twice over the chest of the mouse and tape the harness to the stage plate. Then attach a pulse oximeter to the animal's paw. Then place the environmental chamber on the stage and turn on the heat to maintain the mouse at a physiological temperature.When the animal is in position, bring the 25 by 95 numerical aperture objective lens near the cover slip. In the epifluorescence mode, view the fitzy channel and bring the lung tissue into focus. Next, load a sterile syringe with 100 microliters of the tumor cell suspension, remove the syringe of PBS from the tail vein catheter, and connect the tumor cell syringe.Slowly inject the tumor cells into the tail vein. Then reconnect the PBS syringe to the tail vein catheter and locate the tumor cells throughout the lung tissue via the microscope ocular. Now replace the PBS syringe with a syringe loaded with 100 microliters of rhodamine dextran and slowly inject the dextran into the mouse via the tail catheter.Follow the dextran administration with a 50 microliter, sterile, PBS flush. Then obtain images at each of the previously located tumor cell locations to visualize the flow and integrity of the vasculature. Twenty-four hours post injection, single tumor cells can be visualized interacting with macrophages and monocytes within the vasculature.The stability of the imaging is such that sequential Z-stacks of the field can be acquired and a three dimensional reconstruction can be generated. Here, a five by five mosaic of imaging fields, covering an 890 micron field of view, over 200 and five minutes, at 15 microns below the surface of the lung of a single metastatic lesion, 12 days post injection is shown. Despite the large field of view, the high resolution of the underlying frames, composing the mosaic, enables the capture of subcellular events, such as the mitosis of a single cell, as evidenced by chromosomal separation.Intravascular injection of a high molecular weight, fluorescently labeled dextran, results in the labeling of the vascular lumen;although, unlabeled circulating erythrocytes and leukocytes can occlude the dextran. In the small capillaries of the lung, this occlusion results in a flashing of the dextran signal and a loss of definition in the vascular boundaries. The high spatial stability, provided by this protocol, allows time-averaging of the blood channel without blurring, thus restoring the temporary occlusions.The other signal channels can then be overlayed on the vasculature to provide a clear view of the vessel boundaries. Once mastered, this technique can be completed in about an hour if it's performed properly. While attempting this procedure, it's important to remember to take care not to cut any large blood vessels without first cauterizing them and to avoid touching the lung tissue at all.Following this procedure, the earliest stages of tumor cell dissemination and proliferation can be visualized. After watching this video, you should have a good understanding of how to perform intravital imaging in the intact lung with a vacuum stabilized imaging window. Thanks for watching and good luck with your experiment.

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Research

JoVE Journal

Cancer Research

12.9K Views.  Albert Einstein College of Medicine. The overall goal of this method is to perform long-term, high-resolution, single-cell imaging of the intact lung using a vacuum stabilized imaging window. This method can help answer key questions in the field of cancer research;such as, what interactions secure between stroma and tumor cells during the earliest steps of metastasis. The main advantage of this technique is that the live, intact lung can be theorically visualized at a single-cell resolution for up to 12 hours.Begin by ty...